The long-term objective of this proposal is to uncover mechanisms involved in the regulation of HNSCC pathogenesis and therapy. Human (dihydro)ceramide syntheses 1-6, (dh)CerS1-6, identified as yeast homologues of the longevity assurance gene 1-6 (LASS1-6) regulate the de novo generation of endogenous ceramides with specific fatty acid chain lengths; for example, whereas (dh)CerS1/LASS1 is responsible for the generation of C18-(dihydro)ceramide, (dh)CerS6/LASS6 generates C16-(dihydro)ceramide. These dihydroceramides are then desaturated to ceramides (with their distinct fatty acid chain lengths) by dihydroceramide desaturase (Des). In the cell, these two enzymatic steps (dihydroceramide synthesis and desaturation) occur in the endoplasmic reticulum (ER). Remarkably, our ongoing mechanistic studies reveal that knock down of LASS6/C16-(dihydro)ceramide induces ER stress, which then triggers mitochondrial apoptosis in HNSCC cells. In addition, treatment of HNSCC cells with known ER stress inducers, such as tunicamycin, results in a rapid degradation of LASS6 protein prior to apoptosis. More importantly, increased levels of C16-ceramide via induction of hl_ASS6 expression enhance resistance, and protect HNSCC cells from ER stress and cell death. Collectively, these data suggest a novel hypothesis that LASSS-generated C16-(dihydro)ceramide plays important roles in the regulation of ER homeostasis, such that down-regulation of this pathway mediates a significant component of the ER stress response, which then leads to apoptosis in HNSCC cells. To test this novel hypothesis, three Specific Aims are proposed: 1) Determine the roles of LASS6/C16-(dihydro)ceramide in the regulation of ER stress in HNSCC cells; 2) Identify the mechanisms by which down-regulation of LASS6/C16-(dihydro)ceramide induces ER stress (or a component of the stress), and consequent apoptosis in HNSCC cells; and 3) Establish the in vivo roles and clinical relevance of LASS6/C16-(dihydro)ceramide in HNSCC pathogenesis and/or response to therapy via the regulation of ER stress. Thus, these studies will help determine the roles and mechanisms of action of LASS6/C16- (dihydro)ceramide in the regulation of ER stress in HNSCC cells. Importantly, these results have tremendous implications in unraveling the complexities of ceramide signaling and ER stress, in addition to clear therapeutic implications that will be defined in this proposal.